Chemical approaches for detecting S-nitrosothiols

说明(申请人提供)：一氧化氮是一种细胞信号分子，参与许多生理和病理生理过程。半胱氨酸残基被一氧化氮修饰，即S亚硝化，改变了一系列蛋白质的功能。这一反应代表了一种重要的翻译后修饰，传递了一氧化氮依赖的信号。然而，生物样品中S亚硝化反应的检测和定量仍然是一个挑战，因为S亚硝化产物S-亚硝硫醇(RSNO)不稳定。本课题组最近开发了一系列RSNO的新反应，包括双连接、还原连接和还原消除(生成脱氢丙氨酸)。这些反应选择性地以RSNO为靶标，并将它们转化为稳定的/可检测的产物。在对照实验的基础上，我们假设这些反应可以用来设计检测RSNO的新方法。在本项目中，我们计划追求以下四个具体目标：(1)研究基于双连接的蛋白质S亚硝化的检测方法；(2)研究基于还原连接的蛋白质S亚硝化的检测方法；(3)研究S-亚硝半胱氨酸生成脱氢丙氨酸的方法及其在蛋白质S亚硝化检测中的应用；(4)验证新的RSNO检测方法在生物系统中的应用。这些研究将促进对RSNO的化学和生物学特性的了解，并为研究复杂系统中与S亚硝化相关的NO信号转导提供可能。 公共卫生相关性：蛋白质半胱氨酸残基的S亚硝化在生理学和病理生理学中具有重要作用，而S亚硝化的检测仍是一个挑战。本文提出的研究将为S亚硝化的化学研究提供新的见解，并为生物样品中S亚硝化的检测带来新的技术，这将对生物医学研究产生重大影响。

DESCRIPTION (provided by applicant): Nitric oxide is a cell-signaling molecule involved in a number of physiological and pathophysiological processes. Modification of cysteine residues by nitric oxide, i.e. S- nitrosation, changes the function of a broad spectrum of proteins. This reaction represents an important post-translational modification that transduces nitric oxide- dependent signals. However, the detection and quantification of S-nitrosation in biological samples remains a challenge because of the lability of the S-nitrosation products: S-nitrosothiols (RSNOs). Our group recently developed a series of new reactions for RSNOs, which include bis-ligation, reductive ligation, and reductive elimination (to form dehydroalanine). These reactions selectively target on RSNOs and convert them to stable/detectable products. Based on control experiments, we hypothesize these reactions can be used to design new methods for the detection of RSNOs. In this project, we plan to pursue the following four Specific Aims: (1) to study bis-ligation based methods for detecting protein S-nitrosation; (2) to study reductive ligation based methods for detecting protein S-nitrosation; (3) to study dehydroalanine formation from S-nitrosocysteines and the applications in the detection of protein S- nitrosation; and (4) to validate the applications of new RSNO detection methods in biological systems. These studies will advance the understanding of the chemical and biological properties of RSNOs and permit studies of S-nitrosation-related nitric oxide signal transducation in complex systems. PUBLIC HEALTH RELEVANCE: S-Nitrosation of protein cysteine residues plays important roles in physiology and pathophysiology, while the detection of S-nitrosation is still a challenge. The research proposed here will provide new insights into the chemistry of S-nitrosation and lead to new techniques for the detection of S-nitrosation in biological samples, which should have great impact on biomedical research.